Neuroblastoma (NB), the most frequent extracranial solid tumor in children, causes 15% of pediatric cancer deaths. Greater than 50% of all NB patients present with aggressive metastatic disease. Significantly, the death rate for patients that have metastatic disease at the time of diagnosis remains at ~30-50%, despite aggressive treatment and significant increases in our understanding of the disease. Several genetic abnormalities have been identified in this disease. However, little is known about how these genetic alterations contribute to tumor formation and metastasis, although N-myc amplification correlates with aggressive metastatic disease. Our laboratory discovered that NB cell lines and patients frequently exhibit loss of caspase-8 (C8) expression either by epigenetic mechanisms or in a few cases through gene deletion. We have also shown that the loss of caspase-8 facilitates tumor initiation and enhances metastasis via the prevention of integrin mediated cell death and decreases the responsiveness of tumors to apoptotic stimuli. The studies in this proposal are designed to test the t the hypothesis that caspase-8 plays an important modifier role in N-myc induced NB. We further hypothesize that C8 has both apoptotic and nonapoptotic roles in neuroblastoma formation and metastasis and that modulation of caspase-8 expression and/or activity cooperate with other genetic changes, such as MYCN over-expression in tumor formation. To test this hypothesis we plan to: 1.) develop a mouse model system that recapitulated the increase in MYCN expression and the decrease in caspase-8 expression that is seen in most human patients and to use this system to determine how reducing C8 expression alters the effects of MYCN over-expression on apoptosis, proliferation and survival and 2.) To determine whether caspase-8 plays nonapoptotic roles in tumorigenesis and metastasis in NB tumors that retain C8 expression. These studies will examine whether the apoptotic functions of C8 are abrogated in these cells, by reducing C8 levels below the threshold needed for induction of apoptosis and/or by posttranslational modification that decrease enzymatic activity or alter subcellular localization. We will also delineate that pathways involved in the nonapoptotic functions of C8 in survival and proliferation and determine whether similar decreases in C8 expression or posttranslational modifications are seen in human NB patients. Upon completion of these studies we will have obtained significant new insight into the biology of this complex disease that can be used to develop more targeted treatments and ideally improve the prognosis of NB patients diagnosed with aggressive metastatic disease. In addition, since loss of C8 has been correlated with poor prognosis in medulloblastoma and with relapsed aggressive glioblastoma, which also exhibit N-myc amplification and/or overexpression, the data from these studies may provide insight into the biology of other human tumors. PUBLIC HEALTH RELEVANCE: Neuroblastoma is the most common extracranial solid tumor in childhood, accounting for approximately 7% to10% of pediatric cancers and 15% of all pediatric cancer deaths in patients less than 15 years old. Importantly, >50% of all neuroblastoma patients present with metastatic disease. Despite aggressive treatment 30% to 50% of these patients die. The studies in this proposal are designed to determine the role of caspase-8 and N-myc, two proteins whose expression is altered in neuroblastoma, in tumorigenesis, metastasis and chemotherapeutic responsiveness.